Methods for treating cell proliferative disorders and viral infections

ABSTRACT

The present invention concerns methods for treating cell proliferative diseases, tumors associated with viral infections, and certain viral infections. The disclosed methods use compounds which inhibit heat shock protein 90 proteins. Such methods block Rb negative or deficient cells in the G2/M phase of the cell cycle and rapidly causes their destruction.

DESCIRIPTION

[0001] This application claims the benefit of U.S. ProvisionalApplications Serial No. 60/221,415 filed Jul. 28, 2000 and 60/245,264filed Nov. 2, 2000, both of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The eukaryotic heat shock protein 90s (HSP90s) are ubiquitouschaperone proteins, which bind and hydrolyze ATP. The HSP90 family ofproteins includes four known members: Hsp90 α and β, Grp94 and Trap-1.The roles of HSP90s in cellular functions are not completely understood,but recent studies indicate that HSP90s are involved in folding,activation and assembly of a wide range of proteins, including keyproteins involved in signal transduction, cell cycle control andtranscriptional regulation. For example, researchers have reported thatHSP90 chaperone proteins are associated with important signalingproteins, such as steroid hormone receptors and protein kinases,including many implicated in tumorigenesis, such as Raf-1, EGFR, v-Srcfamily kinases, Cdk4, and ErbB-2 (Buchner J., 1999, TIBS, 24:136-141;Stepanova, L. et al., 1996, Genes Dev. 10:1491-502; Dai, K. et al.,1996, J. Biol. Chem. 271:22030-4).

[0003] In vivo and in vitro studies indicate that without the aid ofco-chaperones HSP90 is unable to fold or activate proteins. For steroidreceptor conformation and association in vitro, HSP90 requires Hsp70 andp60/Hop/Sti1 (Caplan, A., 1999, Trends in Cell Biol., 9: 262-68). Invivo HSP90 may interact with HSP70 and its co-chaperones. Otherco-chaperones associated with HSP90s in higher eukaryotes include Hip,Bag1, HSP40/Hdj2/Hsj1, Immunophillinis, p23, and p50 (Caplan, A. supra).

[0004] Ansamycin antibiotics are natural products derived fromStreptomyces hygroscopicus that have profound effects on eukaryoticcells. Many ansamycins, such as herbimycin A (HA) and geldanamiiycini(GM), bind tightly to a pocket in the HSP90 (Stebbins, C. et al., 1997,Cell, 89:239-250). The binding of ansamycins to HSP90 has been reportedto inhibit protein refolding and to cause the proteasome dependentdegradation of a select group of cellular proteins (Sepp-Lorenzino, L.,et al., 1995, J. Biol. Chem., 270:16580-16587; Whitesell, L. et al.,1994, Proc. Natl. Acad. Sci. USA, 91: 8324-8328).

[0005] The ansamycins were originally isolated on the basis of theirability to revert v-src transformed fibroblasts (Uehara, Y. et al.,1985, J. Cancer Res., 76: 672-675). Subsequently, they were said to haveantiproliferative effects on cells transformed with a number ofoncogenes, particularly those encoding tyrosine kinases (Uehara, Y., etal., 1988, Virology, 164: 294-98). Inhibition of cell growth isassociated with apoptosis and, in certain cellular systems, withinduction of differentiation (Vasilevskaya, A. et al., 1999, CancerRes., 59: 3935-40). A GM derivative is currently in phase I clinicaltrials.

[0006] The use of ansamycins as anticancer agents are described in U.S.Pat. Nos. 4,261,989, 5,387,584 and 5,932,566. The preparation of theansamycin, geldanamiycin, is described in U.S. Pat. No. 3,595,955(incorporated herein by reference).

[0007] The ansamycin-binding pocket in the N-termiinus of Hsp90 ishighly conserved and has weak homology to the ATP-binding site of DNAgyrase (Stebbins, C. et al., supra; Grenert, J. P. et al., 1997, J.Biol. Chem., 272:23843-50). This pocket has been reported to bind ATPand ADP with low affinity and to have weak ATPase activity (Proromou, C.et al., 1997, Cell, 90: 65-75; Panaretou, B. et al., 1998, EMBO J., 17:4829-36). In vitro and in vivo studies are said to indicate thatoccupancy of the pocket by ansamycins alters HSP90 function and inhibitsprotein refolding. At high concentrations, ansamycins have been reportedto prevent binding of protein substrates to HSP90 (Scheibel, T., H. etal., 1999, Proc. Natl. Acad. Sci. USA 96:1297-302; Schulte, T. W. etal., 1995, J. Biol. Chem. 270:24585-8; Whitesell, L., et al., 1994,Proc. Natl. Acad. Sci. USA 91:8324-8328). Alternatively, they have alsobeen reported to inhibit the ATP-dependent release ofchaperone-associated protein substrates (Schleider, C., L. et al., 1996,Proc. Natl. Acad. Sci. USA, 93:14536-41; Sepp-Lorenzino et al., 1995, J.Biol. Chem. 270:16580-16587). In both models, the unfolded substratesare said to be degraded by a ubiquitin-dependent process in theproteasome (Schneider, C., L., supra; Sepp-Lorenzino, supra.)

[0008] In both tumor and nontransformed cells, binding of ansamycins toHSP90 has been reported to result in the degradation of a subset ofsignaling regulators. These include Raf (Schulte, T. W. et al., 1997,Biochem. Biophys. Res. Commun. 239:655-9; Schulte, T. W., et al., 1995,J. Biol. Chem. 270:24585-8), nuclear steroid receptors (Segnitz, B., andU. Gehring. 1997, J. Biol. Chem. 272:18694-18701; Smith, D. F. et al.,1995, Mol. Cell. Biol. 15:6804-12), v-src (Whitesell, L., et al., 1994,Proc. Natl. Acad. Sci. USA 91:8324-8328) and certain transmembranetyrosine kinases (Sepp-Lorenzino, L. et al., 1995, J. Biol. Chem.270:16580-16587) such as EGF receptor (EGFR) and Her2/Neu (Hartmaann,F., et al., 1997, Int. J. Cancer 70:221-9; Miller, P. et al., 1994,Cancer Res. 54:2724-2730; Mimnaugh, E. G., et al., 1996, J. Biol. Chem.271:22796-801; Schnur, R. et al., 1995, J. Med. Chem. 38:3806-3812). Theansamycin-induced loss of these proteins is said to lead to theselective disruption of certain regulatory pathways and results ingrowth arrest at specific phases of the cell cycle (Muise-Heimericks, R.C. et al., 1998, J. Biol. Chem. 273:29864-72).

[0009] Cyclin D in complex with Cdk4 or Cdk6 and cyclin E-Cdk2phosphorylate the protein product of the retinoblatoma gene, Rb.Researchers have reported that the protein product of the Rb gene is anuclear phosphoprotein, which arrests cells during the G₁ phase of thecell cycle by repressing transcription of genes involved in the G₁ to Sphase transition (Weinberg, R. A., 1995, Cell, 81:323-330).Dephosphorylated Rb is said to inhibit progression through late G₁, inpart, through its interaction with E2F transcription family members,which ultimately represses the transcription of E2F target genes (Dyson,N., 1998, Genes Dev., 12: 2245-2262). Progressive phosphorylation of Rbby the cyclin-dependent kinases in mid to late G₁ leads to dissociationof Rb from Rb-E2F complexes, allowing the expression of E2F target genesand entry into the S phase.

[0010] The retinoblastoma gene product is mutated in several tumortypes, such as retinoblastoma, osteosarcoma and small-cell lung cancer.Research also indicates that in many additional human cancers thefunction of Rb is is disrupted through neutralization by a bindingprotein, (e.g., the human papilloma virus-E7 protein in cervicalcarcinoma; Ishiji, T, 2000, J Dermiiatol., 27: 73-86) or deregulation ofpathways ultimately responsible for its phoshorylation. Inactivation ofthe Rb pathway often results from pertubation of p16INKa, Cyclin D1, andCdk4.

[0011] The retinoblastoma gene product, besides being a target of humanpapilloma E7 protein, is also the target of other oncogenic viral geneproducts. For example, studies indicate that the simian virus 40 large Tantigen inactivates the Rb family of proteins, including Rb, p107, andp130 (Zalvide, J. H. et al., 1998, Mol. Cell. Biol., 18: 1408-1415).Research also indicates that transformation by adenovirus requires E1Abinding to Rb (Egan, C. et al., 1989, Oncogene, 4:383-388).

[0012] Scientists estimate that over 70 types of papilloma virusesinfect humans (HPV) (Sasagawa, T. et al., 1996, Clinical Diag. Lab.Immunol, 3: 403-410). Of these several are associated with malignanciesof humans, particularly cervical cancers (Bosch et al., 1995, J. Natl.Cancer Inst., 87:796-802). Recent evidence also implicates HPV in somehead and neck cancers. Several types of HPV are associated with anintermediate to high risk of malignancies (types 16, 18, 31, 33, 35, 45,and 56) (Sasagawa, T., et al., supra). In infections with these HPV, theviral genome integrates into the genome of the infected cell withsubsequent expression of transforming genes E6 and E7. Data indicatethat the products of these genes may promote malignant transformation byaltering the functions of two cellular tumor suppressor proteins (p53and Rb). E6 causes the proteolytic degradation of p53 (Scheffiner, M. etal., 1990, Cell, 63: 1129-1136. E7 complexes with Rb causing its releasefrom transcription factor E2F, leading to the activation of genesinvolved in cell proliferation (Dyson, N. et al., 1988, Science, 243:934-937.).

[0013] Most cancer therapies are not successful with all types ofcancers. For example, solid tumor types ultimately fail to respond toeither radiation or chemotherapy. There remains a need for cancertreatments which target specific cancer types. The present inventionsatisfies these needs and provides related advantages as well. Thepresent invention provides novel methods for treating cell proliferativedisorders and viral infections associated with retinoblastoma negativeor deficient cells.

SUMMARY OF THE INVENTION

[0014] The present invention relates to methods useful for the treatmentof an animal, preferably a mammal, that has a cell proliferativedisorder or viral infection associated with Rb negative or deficientcells. One such method comprises administering an effective amount of apharmaceutical composition that comprises a pharmaceutically acceptablecarrier and a compound that binds to the N-terminal pocket of heat shockprotein 90 to cells that are Rb negative or Rb deficient. In a preferredembodiment the HSP90 binding compound is an ansamycin. In a particularlypreferred embodiment, the ansamycin is17-allylamino-(17)-demethoxygeldanamycin (17-AAG).

[0015] The present invention further provides methods of destroyingcells that are deficient in the retinoblastoma gene product. In one suchembodiment, the method comprises administering an effective amount of acompound that binds to the N-terminal pocket of HSP90 to cells that areRb negative or Rb deficient. In one embodiment, the HSP90 bindingcompound is an ansamycin. In a particularly preferred embodiment, theansamycin is 17-AAG.

[0016] In another embodiment, the invention provides a method ofdestroying Rb negative or Rb deficient cells, comprising administeringan effective amount of a compound that binds to the N-terminal pocket ofHSP90 selected from the group consisting of herbimycin, geldanamycin,and 17-AAG, radicicol or synthetic compounds that bind into theN-terminal pocket of HSP90 which is the ATP-binding site of HSP90.

[0017] The method can further comprise treating a mammal in combinationwith other therapies. Other such therapies include, but are not limitedto, chemotherapy, surgery, and/or radiotherapy.

[0018] By means of the invention, a method of destroying cells which areRb negative or Rb deficient is provided. The invention provides a meansto treat cell proliferative disorders, tumors associated with viralinfections and certain viral infections associated with an Rb negativephenotype. These and other advantages of the present invention will beappreciated from the detailed description and examples set forth below.The detailed description and examples enhance the understanding of theinvention, but are not intended to limit the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1 shows differential cell cycle effects of Herbimycin onRb-wild type (A) and Rb-negative cells (B). (A) MCF7 and Colo 205; (B)MB-MDA 468 and BT 549

[0020]FIG. 2 shows levels of mitotic cyclin expression and associatedkinase activities in Herbimycin arrested MB-MD 468 cells. FIG. 2(A)shows a western blot using anti-cyclin A and also shows an in vitrokinase assay of immunoprecipitates isolated with anti-cyclin A. FIG.2(B) shows a western blot using anti-cyclin B1 antibodies and also showsan iii vitro kinase assay of immunoprecipitates isolated withanti-cyclin B1.

[0021]FIG. 3 shows Rb-wild-type cells complete mitosis in the presenceof HA after arrest with aphidicolin (FIG. 3A). FIG. 3B shows that, afterrelease from aphidicolin, Rb-negative MB-MDA 468 cells arrested in thenext mitosis

[0022]FIG. 4A and B shows that HA induces mitotic arrest and not G,arrest in primary cells expressing HPV 16 E6 and E7.

[0023]FIG. 5 shows the effect of HA on Rb-negative cells transfectedwith the Rb gene. FIG. 5A shows a western blot analysis of Rb expressionin MB-MDA 468, 468-7 and 468-19 FIGS. 5B-D show that introduction of theRb gene abrogates HA-induced mitotic arrest in MB-MDA 468 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention concerns the surprising discovery thatansamycins cause Rb negative or Rb deficient cells to undergo mitoticarrest followed by rapid programmed cell death. This is in contrast toansamycin treatment of cells containing wild-type levels of Rb, whichcauses cells to arrest in GI of the cell cycle followed, in some cases,by differentiation and apoptosis. The induction of mitotic arrest byansamycins in Rb negative or Rb deficient cells, which rapidly leads toprogrammed cell death, is a phenomenon confined to cells with defectiveRb function. Mitosis is unaffected in normal cells with wild-type Rb.Thus, the present invention will aid in the treatment of cellproliferative disorders which are associated with Rb negative or Rbdeficient cells, such as small-cell lung cancers, retinoblastoma,osteosarcoma, certain breast cancers, prostate cancer, bladder cancer,hepatocarcinoma, certain viral infections, and virally induced tumors,including those caused by human papilloma viruses, such as cervicalcarcinoma.

[0025] As used in the specification and claims of this application, theterm “Rb deficient” describes several types of cells, including cellswhich produce no detectable amounts of a functional Rb protein. Suchcells are referred to herein as “Rb negative” cells. Cells which are Rbdeficient may be cells which do not contain a functional Rb gene. Cellswhich are Rb deficient may also be cells that can encode an Rb protein,but in which the protein does not function properly or is produced atlower than normal level. An Rb deficient phenotype can also occur due tothe perturbation of the pathway which ultimately results inphosphorylation of the Rb protein, for example, perturbation ofp16INK4a, Cyclin D1, or Cdk4, and cells with such a perturbation are Rbdeficient cells.

[0026] As used in the specification and claims of this application, theterm “HSP90” refers to the family of HSP90 heat shock proteins. Thus,this term encompasses Hsp90 α and Hsp90β, Grp94 and Trap-1. The HSP90heat shock proteins each possess a characteristic pocket located nearthe N-terminal end of the protein to which ATP and ADP bind. This is thesame pocket which has been shown to bind to ansamycin antibiotics. Thispocket is referred to herein as “the N-terminal pocket of HSP90”.

[0027] Although the precise mechanisms are not yet understood, thepresent application makes use of compositions that bind to theN-terminal pocket of HSP90 in a manner that results in an alteration ofthe function of HSP90. As used in the specification and claims of thisapplication, this alteration of function is referred to as “inhibitionof HSP90 function”. In accordance with the present invention, thisinhibition occurs upon adminstration of HSP90 binding compounds, such asansamycins, and results in arrest of Rb negative or deficient cells inmitosis. Such cells uniformly die through apoptotic mechanisms. Thisnovel mechanism of destroying cells that are Rb negative or deficientprovides a means to specifically treat cell proliferative disorders andcertain viral infections associated with cells that are Rb negative ordeficient.

[0028] The destruction of Rb negative or deficient cells can occur withless cytotoxicity to normal cells or tissues. For example, when cellswhich contain a normal Rb gene product are treated with HSP90inhibitors, those cells arrest in G₁ of the cell cycle and, in somecases, may differentiate and die. However, cells which are Rb negativeor deficient uniformly die when treated with HSP90 inhibitors. Further,such cells will be more susceptible to other agents or radiationtreatments and will require lower doses of drug for killing than cellswith wild-type retinoblastoma gene product. Studies indicate that theG₂/M phase of the cell cycle is the most radiosensitive phase of thecell cycle (Sinclair, W. K, 1968, Radiat. Res., 33:620).

[0029] In one embodiment of the invention, the IC₅₀ of the HSP90inhibitor used in the instant methods to destroy cells which are Rbnegative of Rb deficient is lower than the IC₅₀ against similar cellswhich are not Rb negative or deficient. Preferably the IC₅₀ is 5-foldlower, more preferably 10-fold lower, still further 20-fold lower, andmost preferably 30- to 50-fold lower when compared to similar cellscontaining wild-type Rb.

[0030] As used herein “IC₅₀” is defined as the concentration of an HSP90inhibitor required to achieve killing of 50% of cells.

[0031] The term “effective amount” as used herein, means an amount of acompound utilized in the methods of the present invention which iscapable of providing a therapeutic effect. The specific dose of compoundadministered according to this invention to obtain therapeutic and/orprophylactic effects will, of course, be determined by the particularcircumstances surrounding the case, including, for example, the compoundadministered, the route of administration, the condition being treatedand the individual being treated. A typical daily dose (administered insingle or divided doses) will contain a dosage level of from about 0.01mg/kg to about 50 mg/kg of body weight of an active compound of thisinvention. Preferred daily doses generally will be from about 0.05 mg/kgto about 20 mg/kg and ideally from about 0.1 mg/kg to about 10 mg/kg.

[0032] The preferred therapeutic effect of the methods of the instantinvention, with respect to cell proliferative disorders is theinhibition, to some extent, of growth of cells causing or contributingto a cell proliferative disorder. A therapeutic effect relieves to someextent one or more of the symptoms of a cell proliferative disorder. Inreference to the treatment of a cancer, a therapeutic effect refers toone or more of the following: 1) reduction in the number of cancercells; 2) reduction in tumor size; 3) inhibition (i.e., slowing to someextent, preferably stopping) of cancer cell infiltration into peripheralorgans; 3) inhibition (i.e., slowing to some extent, preferablystopping) of tumor metastasis; 4) inhibition, to some extent, of tumorgrowth; and/or 5) relieving to some extent one or more of the symptomsassociated with the disorder.

[0033] In reference to the treatment of a cell proliferative disorderother than a cancer, a therapeutic effect refers to either: 1) theinhibition, to some extent, of the growth of cells causing the disorder;2) the inhibition, to some extent, of the production of factors (e.g.,growth factors) causing the disorder; and/or 3) relieving to some extentone or more of the syiiptoms associated with the disorder.

[0034] With respect to viral infections, the preferred therapeuticeffect is the inhibition of a viral infection. More preferably, thetherapeutic effect is the destruction of cells which contain the virus.

[0035] The methods of this invention are useful for inhibiting cellproliferative diseases associated with Rb negative or Rb deficient, forexample, retinoblastoma, osteosarcoma, breast cancers, bladder cancer,prostate cancer, renal carcinoma, cancers associated with viralinfections, such as cervical cancers associated with human papillomavirus, and small-cell lung cancer. Additionally, the methods of theinvention are useful for the treatment of certain viral infections whichresult in an Rb negative phenotype, such as human papilloma virus.

[0036] “Cell proliferative disorders” refer to disorders whereinunwanted cell proliferation of one or more subset(s) of cells in amulticellular organism occurs, resulting in harm, for example, pain ordecreased life expectancy to the organism. Cell proliferative disordersinclude, but are not limited to, tumors, benign tumors, blood vesselproliferative disorders, autoimmune disorders and fibrotic disorders.

[0037] The methods of the present invention may be used on mammals,preferably humans, either alone or in combination with other therapiesor methods useful for treating a particular cell proliferative disorderor viral infection.

[0038] The use of the present invention is facilitated by firstidentifying whether the cell proliferation disorder or viral infectionis accompanied by cells which contain altered expression of the Rb geneproduct. Once such disorders are identified, patients suffering fromsuch a disorder can be identified by analysis of their symptoms byprocedures well known to medical doctors. Such patients can then betreated as described herein.

[0039] The determination of whether the cell proliferation disorder isassociated with an altered expression of the Rb gene product call becarried out by first determining the protein expression of Rb in theappropriate cells isolated from a mammal suspected of having a cellproliferative disorder or viral infection. For example, in the case ofsmall-cell lung cancer, the protein expression of Rb determined fromcells isolated from a mammal suspected of having small cell lung cancercan be compared to the appropriate cells isolated from a disease freemammal. Rb expression and/or mutations can be measured using methodswell known in the art, including, but not limited to,immunohistochemistry, Southern blot analysis, and Northern blotanalysis. The use of immunohistochemistry (e.g., Western blot analysis)to determine Rb expression is described by Higashiyam M et al., 1994,Oncogene, 51: 544-51, and Kohn G. J et al., 1997, J. Gasroeiiterol.Hepatol., 12: 198-203, both of these references are incorporated hereinby reference in their entireties. The use of Southern blot analysis todetermine defects in the Rb gene is demonstrated by Presti J. C. Jr. etal., 1996, Anticancer Res., 16:549-56, which is incorporated herein byreference in its entirety. The determination of Rb mRNA using Northernblot analysis is demonstrated by Rygaard K. et al., 1990, Cancer Res.,50: 5312-7, which is incorporated by reference herein in its entirety.If the analysis indicates that there is altered Rb expression, thepatient is a candidate for treatment using the methods described herein.

[0040] In the case of cell proliferative disorders arising due tounwanted proliferation of non-cancer cells, the level of the Rb geneproduct is compared to that level occurring in the general population(e.g., the average level occurring in the general population of peopleor animals excluding those people or animals suffering from a cellproliferative disorder). If the unwanted cell proliferation disorder ischaracterized by an abnormal level of Rb than occurring in the generalpopulation, then the disorder is a candidate for treatment using themethods described herein.

[0041] Methods to determine HPV association of with cervical cancer aredescribed in Sasagawa, T. et al., supra, which is incorporated herein byreference.

[0042] Cell proliferative disorders, including those referenced aboveare not necessarily independent. For example, fibrotic disorders may berelated to, or overlap with, blood vessel disorders. Additionally, forexample, atherosclerosis (which is characterized herein as a bloodvessel disorder) is associated with the abnormal formation of fibroustissue.

[0043] A cancer cell refers to various types of malignant neoplasms,most of which can invade surrounding tissues, and may metastasize todifferent sites, as defined by Stedman's Medical Dictionary 25th edition(Hensyl ed. 1990).

[0044] The formation and spreading of blood vessels, or vasculogenesisand angiogenesis respectively, play important roles in a variety ofphysiological processes such as embryonic development, wound healing andorgan regeneration. They also play a role in cancer development. Bloodvessel proliferation disorders refer to angiogenic and vasculogenicdisorders generally resulting in abnormal proliferation of bloodvessels. Examples of such disorders include restenosis, retinopathies,and atherosclerosis.

[0045] As noted above, other such proliferative diseases can beidentified by standard techniques, and by determination of the efficacyof action of the compounds described herein.

[0046] A. Rb Negative or Deficient Cells Arrest in Mitosis afterTreatment with Ansamycins

[0047] Rb negative or deficient cells treated with ansamycin orradicicol were discovered to contain a bipolar spindle and elevatedcyclin B1-associated kinase activity. However, chromosomal alignment wasdisorganized, with chromosomes scattered along the length of thespindle. The presence of paired chromosomes at the poles led to theconclusion that HA-treated cells had arrested in prometaphase as aresult of failure of chromosomes to align into a metaphase plate. Thisarrest was dependent on the absence of Rb as introduction of wild-typeRB allowed progression through mitosis in the presence of drug. Whentreated with ansamycins in S phase, Rb-negative cells blocked in thesubsequent mitosis whereas Rb-wild type cells progressed through mitosisand arrested in G. Thus, Rb is required for completion of mitosis whenHsp90 function is inhibited.

[0048] In 12 tumor cell lines examined, ansamycin treatment causedgrowth arrest in GI (FIG. 1A). This arrest was accompanied by a rapiddecline in D-cyclin-associated kinase activity and hypophosphorylationof Rb, suggesting that ansamycins affect G₁ via a cyclin D-relatedpathway (Srethapakdi, M., F. Liu, R. Tavorath, and N. Rosen, 2000,Cancer Res. 60: 3940-6). These effects were elicited by three differentansarnycins, HA, GM and its derivative,17-allylamino-(17)demethoxygeldanamycin (17-AAG), differing only inregard to potency. Although these experiments were done, for the mostpart, with HA, it will be understood that similar effects can beobtained using other ansamycins, which bind to the HSP90 pocket, such asthe benzoquinone ansamycins, including, but not limited to,geldanamycin, geldanamycin derivatives, such as 17-AAG, herbimycin, andmacbecins, or other compounds which bind to the HSP 90 pocket, such asradicicol. To determine if ansamycins disrupted G₁, progression byinhibiting the cyclin D-Rb pathway, their effects were examined in celllines lacking functional Rb. Rb is the only known substrate of cyclinD-associated kinases (Baldin, V., et al., 1993, Genes Dev. 7:812-21;Ewen, M. E. et al., 1993, Cell 73:487-97; Kato, J., H. et al., 1993,Genes Dev. 7:331-42; Matsushime, H., et al., 1992, Cell 71:323-34;Matsushime, H., D. et al., 1994, Mol. Cell. Biol. 14:2066-76; Meyerson,M., and E. Harlow, 1994, Mol. Cell. Biol. 14:2077-86; Quelle, D. E., etal., 1993, Genes Dev. 7:1559-1571; Guan, K.-L., et al., 1994, Genes Dev.8:2939-52; Koh, J. et al., 1995, Nature 375:506-10; Lukas, J. et al.,1995, Mol. Cell. Biol. 15:2600-11; Lukas, J., H. et al., 1994, J. CellBiol. 125:625-38; Lukas. J., D. et al., 1995, Nature 375:503-6; Medema,R. H. et al., 1995, Proc. Natl. Acad. Sci. USA, 92:6289-93). In tumorcell lines with mutated Rb (MB-MDA 468, BT-549, DU145 and DU4475) HAtreatment failed to induce a G₁ block but instead led to an accumulationof cells with 4n DNA content (FIG. 1B).

[0049] To determine if HA treatment caused Rb-negative cells to arrestin G₂ or mitosis, mitotic index was determined with bisbenzimidestaining and mitosis was scored by the presence of condensedchromosomes. In MB-MDA 468 cells, in which the mitotic index of thecontrol population was 5-10%, 60-70% of HA-treated cells were inmitosis. Thus, in the absence of Rb function, HA treatment resulted inmitotic arrest.

[0050] To further define the nature of the HA-induced mitotic defect,cells were triple-stained with bisbenzimide, anti-α-tubulin antibodiesand anti-centromere autoimmune serum (ACA/CREST). α-tubulin stainingrevealed that arrested cells contained bipolar spindles, demonstratingthat HA does not interfere with spindle formation. Examination ofcluomosomnal distribution by bisbenzamide and ACA/CREST staining,however, showed that in most cells, chromosomes localized both to thepoles and within the spindle.

[0051] Without being bound to any particular theories, the observedaccumulation of chromosomes at the poles is consistent with either anarrest in prometaphase due to failure of chromosomes to align into ametaphase plate or to an abnormal anaphase with impaired sisterchromatid segregation. ACA staining, however, revealed pairedcentromeres on chromosomes at the poles, indicating that they wereundisjoined sister chromatids (FIG. 3B). In 77 chromosomes localized tothe poles, 87% scored as double dots for ACA staining. This demonstratesthat accumulation of chromosomes at the poles did not result frompremature or incomplete segregation but rather, failure of pairedchromatids to congress to the spindle equator. These data show thatHA-treated cells are arrested in prometaphase and that, in Rb-negativecells, HA induces mitotic arrest by interfering with chromosomalalignment.

[0052] To further distinguish between prometaphase and anaphase, theexpression and -associated kinase activities of the mitotic cyclins wereassessed. Levels of cyclin A-associated kinase activity begun to declinein prometaphase while cyclin B associated kinase activity remainselevated until anaphase (Furuno, N., N. den Eizen, and J. Pines, 1999,J. Cell Biol. 147:295-306; Townsley, F. M., and J. V. Ruderman, 1998,Trends Cell Biol. 8:238-244; Zachariae, W., and K. Nasmyth, 1999, GenesDev. 13:2039-58). As the mitotic index in the HA-blocked population isonly 60-70%, mitotically arrested cells were enriched by using only theloosely adherent population in which the mitotic index was greater than90%. Cyclin B1-associated kinase activity was elevated 5-fold inHA-treated cells when compared to control and was comparable to thatseen in nocodazole-arrested cells (FIG. 2B). In parallel with kinaseactivity, cyclin B1 protein expression was also increased in HA-treatedcells (FIG. 2B). In contrast, cyclin A expression and its associatedkinase activity were slightly lower in both HA and nocodazole-arrestedcells compared to that in control cells (FIG. 2A). Thus, HA inducesarrest at a point before early anaphase and after prophase whenproteolysis of cyclin A but not cyclin B1, has begun. This result showsthat arrest occurs in prometaphase of mitosis.

[0053] Cells with Wild-Type RB Traverse Mitosis in the Presence of HA

[0054] The HA-induced mitotic block was observed, surprisingly, only incells lacking wild-type Rb. HA likely causes the degradation of mitoticregulators more slowly than it affects the expression of G₁ regulators.The absence of Rb would abrogate the effects on G₁ and expose themitotic phenotype. The addition of HA to Rb-negative cells blocked in Sphase, then, would fail to cause arrest in the mitosis immediatelyfollowing drug addition. To demonstrate this, Colo 205 cells and MB-MDA,468 cells were arrested in G₁/S with aphidicolin and subsequentlyreleased from block in the presence of either HA or DMSO. In thepresence of HA, Rb-wild type Colo 205 cells progressed through G2 andmitosis and were arrested in the next G₁ (FIG. 3A). In contrast,following release from aphidicolin, Rb-negative MB-MDA 468 cellsarrested within 12 hours in the next mitosis (FIG. 3B). Thus, cellscontaining Rb are able to progress normally through mitosis in thepresence of HA while those lacking Rb function are not.

[0055] HA Induces M and not G, Arrest in Primary Cells Expressing HPV 16E6 and E7

[0056] With regard to whether the above observations could result frommutations in other genes that complement with Rb mutations to causetransformation, the cell cycle effects of HA were examined in primaryhuman foreskin keratinocytes (HFK) expressing human papilloma virus-16(HPV-16) E6 and E7. These viral oncogenes functionally inactivate p53and Rb, respectively. Introduction of both E6 and E7 was necessary asloss of Rb function in a p53 wild-type background has been shown topredispose cells to undergo apoptosis (Jones, D. L., D. A. Thompson, andK. Munger, 1997, Virology, 239:97-107; Pan, H., and A. E. Griep, 1994,Genes Dev. 8:1285-99; White, A. E., E. M. Livanos, and T. D. Tlsty,1994, Genes Dev. 8:666-77). While HA caused the majority of primary HFKcells (FIG. 4A) to accumulate in G₁, E6/E7 transfectants arrested with4n DNA content (FIG. 4B). These results provide further evidence thatthe cell cycle response to the HA is dictated by the status of Rb andmoreover, that Rb is required for mitotic traversal following drugexposure. The loss of p53 function alone is not sufficient for mitoticblock as the multiple p53-negative/Rb-positive cell lines that have beentested successfully traverse mitosis in the presence of ansamycins.

[0057] Introduction of Rb into Rb-Negative Cells Allows Progressionthrough M in the Presence of HA

[0058] The discovery that addition of HA to cells in S phase inducesmitotic arrest in Rb negative MB-MDA 468,cells but not Rb-wild type Colo205 cells indicates that Rb permits progression through mitosis underthese conditions. To test this, wild type Rb was reintroduced into thecell line MB-MDA 468. A low transfection efficiency was seen, possiblybecause elevated expression of Rb inhibits cell growth. Five positiveclones were ultimately obtained. These transfectants expressed lowerlevels of Rb when compared to Rb-wild-type tumor cell lines. Two stablytransfected clones expressing different levels of Rb (468-7 and 468-19)were chosen for analysis (FIG. 5A). FACS analysis of logarithmicallygrowing populations revealed that Rb expression in these clones did notalter the cell cycle distribution, though the cells had slightly longerdoubling times. When treated with HA, control transfectants accumulatedwith 4n DNA content. In contrast, the drug had no effect on G₂/M in theRb-transfectants and instead caused an increase in G₁. Furthermore, whenreleased from aphidicolin block into HA, both clone 468-7 and 468-19cycled through mitosis and entered G₁, (FIGS. 5C & D). In contrast, whentreated with HA after aphidicolin block, MB-MDA 468 cells failed toreach G₁ and arrested in mitosis by 12 hours (FIG. 5D). The amount ofcell death induced by ansamycins was comparable in the Rb-transfectedand untransfected cells. Thus, in the Rb-transfectants, the appearanceof a higher percentage of cells in G₁ does not result from increasedapoptosis of cells in G₂/M. As these cell lines differ only in Rbstatus, this finding demonstrates that Rb expression alone is sufficientto allow progression through mitosis in the presence of HA.

[0059] Inhibition of Hsp90 with Radicicol Induces Mitotic Arrest inMB-MDA 468 Cells

[0060] HA binds to Hsp90 but may have other effects that relate to itschemical properties. Treatment with GM and 17-AAG generated the sameRb-dependent cell cycle profiles and mitotic phenotype as observed withHA. Radicicol is a non-ansamycin natural product that has been shown tobind to the N-terminal Hsp90 pocket (Schulte, T. et al., 1999, Mol.Enidocrinol. 13:1435-1448; Schulte, T. et al., 1998, Cell StressChaperones 3:100-8) and to induce the degradation of the same spectrumof proteins affected by ansamycins (Soga, S., et al., 1998, J. Biol.Chest. 273:822-828). Radicicol treatment induced G₁ arrest inRb-positive cell lines, Colo2O5 and MCF7, but failed to arrestRb-negative MDA-468 cells in G₂, and instead, like ansamycins, caused anaccumulation of cells with 4n DNA content. Radicicol-arrested MDA 468cells also displayed chromosomes localized to the poles as well asstrewn along the spindle.

[0061] B. Administration and Pharmaceutical Compositions

[0062] The compounds utilized in the methods of the instant inventionmay be administered either alone or in combination with pharmaceuticallyacceptable carriers, excipients or diluents, in a pharmaceuticalcomposition, according to standard pharmaceutical practice. Thecompounds can be administered orally or parenterally, including theintraventous, intramuscular, intraperitoneal, subcutaneous, rectal andtopical routes of administration.

[0063] The pharmaceutical compositions used in the methods of theinstant invention can contain the active ingredient in a form suitablefor oral use, for example, as tablets, troches, lozenges, aqueous oroily suspensions, dispersible powders or granules, emulsions, hard orsoft capsules, or syrups or elixirs. Compositions intended for oral usemay be prepared according to any method known to the art for themanufacture of pharmaceutical compositions and such compositions maycontain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients which are suitable forthe manufacture of tablets. These excipients may be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,such as microcrystalline cellulose, sodium crosscarmellose, corn starch,or alginic acid; binding agents, for example starch, gelatin,polyvinyl-pyrrolidone or acacia, and lubricating agents, for example,magnesium stearate, stearic acid or talc. The tablets may be uncoated orthey may be coated by known techniques to mask the unpleasant taste ofthe drug or delay disintegration and absorption in the gastrointestinaltract and thereby provide a sustained action over a longer period. Forexample, a water soluble taste masking material such ashydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delaymaterial such as ethyl cellulose, cellulose acetate butyrate may beemployed.

[0064] Formulations for oral use may also be presented as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredient is mixed withwater soluble carrier such as polyethyleneglycol or an oil medium, forexample peanut oil, liquid paraffin, or olive oil.

[0065] Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

[0066] Oily suspensions may be formulated by suspending the activeingredient in a vegetable oil, for example arachis oil, olive oil,sesame oil or coconut oil, or in mineral oil such as liquid paraffin.The oily suspensions may contain a thickening agent, for examplebeeswax, hard paraffin or cetyl alcohol. Sweetening agents such as thoseset forth above, and flavoring agents may be added to provide apalatable oral preparation. These compositions may be preserved by theaddition of an antioxidant such as butylated hydroxyanisol oralpha-tocopherol.

[0067] Dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water provide the activeingredient in admixture with a dispersing or wetting agent, suspendingagent and one or more preservatives. Suitable dispersing or wettingagents and suspending agents are exemplified by those already mentionedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present. These compositions may bepreserved by the addition of an anti-oxidant such as ascorbic acid.

[0068] The pharmaceutical compositions used in the methods of theinstant invention may also be in the form of an oil-in-water emulsions.The oily phase may be a vegetable oil, for example olive oil or arachisoil, or a mineral oil, for example liquid paraffin or mixtures of these.Suitable emulsifying agents may be naturally-occurring phosphatides, forexample soy bean lecithin, and esters or partial esters derived fromfatty acids and hexitol anhydrides, for example sorbitan monooleate, andcondensation products of the said partial esters with ethylene oxide,for example polyoxyethylene sorbitan monooleate. The emulsions may alsocontain sweetening, flavoring agents, preservatives and antioxidants.

[0069] Syrups and elixirs may be formulated with sweetening agents, forexample glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative, flavoring andcoloring agents and antioxidant.

[0070] The pharmaceutical compositions may be in the form of a sterileinjectable aqueous solutions. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution.

[0071] The sterile injectable preparation may also be a sterileinjectable oil-in-water microemulsion where the active ingredient isdissolved in the oily phase. For example, the active ingredient may befirst dissolved in a mixture of soybean oil and lecithin. The oilsolution then introduced into a water and glycerol mixture and processedto form a microemulation.

[0072] The injectable solutions or microemulsions may be introduced intoa patient's blood-stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

[0073] The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

[0074] The HSP90 inhibitors used in the methods of the present inventionmay also be administered in the form of a suppositories for rectaladministration of the drug. These compositions can be prepared by mixingthe inhibitors with a suitable nonirritating excipient which is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials includecocoa butter, glycerinated gelatin, hydrogenated vegetable oils,mixtures of polyethylene glycols of various molecular weights and fattyacid esters of polyethylene glycol.

[0075] For topical use, creams, ointments, jellies, solutions orsuspensions, etc., containing an HSP90 inhibitor can be used. (As usedherein, topical application can include mouth washes and gargles.)

[0076] The compounds used in the methods of the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles and delivery devices, or via transdermal routes, using thoseforms of transdermal skin patches well known to those of ordinary skillin the art. To be administered in the form of a transdermal deliverysystem, the dosage administration will, of course, be continuous ratherthan intermittent throughout the dosage regimen.

[0077] The HSP90 inhibitors used in the instant invention may also beco-administered with other well known therapeutic agents that areselected for their particular usefulness against the condition that isbeing treated. For example, the instant compounds may be useful incombination with known anti-cancer and cytotoxic agents. Similarly, theinstant compounds may be useful in combination with agents that areeffective in the treatment and prevention of certain viral infections orother conditions associated with an Rb negative phenotype. The instantcompounds may also be useful in combination with other inhibitors ofparts of the signaling pathway that links cell surface growth factorreceptors to nuclear signals initiating cellular proliferation.

[0078] The methods of the present invention may also be useful withother agents that inhibit angiogenesis and thereby inhibit the growthand invasiveness of tumor cells, including, but not limited to VEGFreceptor inhibitors, including ribozymes and antisense targeted to VEGFreceptors, angiostatin and endostatin.

[0079] Examples of an antineoplastic agents, which can be used incombination with the methods of the present invention include, ingeneral, alkylating agents, anti-metabolites; epidophyllotoxin; anantineoplastic enzyme; a topoisomerase inhibitor; procarbazine;mitoxantrone; platinum coordination complexes; biological responsemodifiers and growth inhibitors; hormonal/anti-hormonal therapeuticagents and haematopoietic growth factors.

[0080] Example classes of antineoplastic agents includes for example,the anthracycline family of drugs, the vinca drugs, the mitomycins, thebleomycins, the cytotoxic nucleosides, the epothilones, discodermolide,the pteridine family of drugs, diynenes and the podophyllotoxins.Particularl useful members of those classes include, for example,carminomycin, daunorubicin, aminopterin, methotrexate, methopterin,dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil,6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin orpodophyllotoxin derivatives such as etoposide, etoposide phosphate orteniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine,leurosine, paclitaxel and the like. Other useful antineoplastic agentsinclude estramustine, carboplatin, cyclophosphamide, bleomycin,gemcitibine, ifosamide, melphalan, hexamethyl melamine, thiotepa,cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase,camptothecin, CPT-11, topotecan, ara-C, bicalutamide, flutamide,leuprolide, pyridobenzoindole derivatives, interferons and interleukins.

[0081] When a HSP90 inhibitor used in the methods of the presentinvention is administered into a human subject, the daily dosage willnormally be determined by the prescribing physician with the dosagegenerally varying according to the age, weight, and response of theindividual patient, as well as the severity of the patient's symptoms.

[0082] In one exemplary application, a suitable amount of a HSP90inhibitor is administered to a mammal undergoing treatment for cancer.Administration occurs in an amount of each type of inhibitor of betweenabout 0.1 mg/kg of body weight to about 60 mg/kg of body weight per day,preferably of between 0.5 mg/kg of body weight to about 40 mg/kg of bodyweight per day. A particular therapeutic dosage that comprises theinstant composition includes from about 0.01 mg to about 1000 mg of aHSP90 inhibitor. Preferably, the dosage comprises from about 1 mg toabout 1000 mg of a HSP90 inhibitor.

[0083] Preferably, the pharmaceutical preparation is in unit dosageform. In such form, the preparation is subdivided into unit dosescontaining appropriate quantities of the active component, e.g., aneffective amount to achieve the desired purpose.

[0084] The quantity of active compound in a unit dose of preparation maybe varied or adjusted from about 0.1 mg to 1000 mg, preferably fromabout 1 mg to 300 mg, more preferably 10 mg to 200 mg, according to theparticular application.

[0085] The actual dosage employed may be varied depending upon therequirements of the patient and the severity of the condition beingtreated. Determination of the proper dosage for a particular situationis within the skill of the art. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small amounts until the optimumeffect under the circumstances is reached. For convenience, the totaldaily dosage may be divided and administered in portions during the dayif desired.

[0086] The amount and frequency of administration of the HSP90inhibitors used in the methods of the present invention and ifapplicable other chemotherapeutic agents and/or radiation therapy willbe regulated according to the judgment of the attending clinician(physician) considering such factors as age, condition and size of thepatient as well as severity of the disease being treated.

[0087] The chemotherapeutic agent and/or radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the chemotherapeutic agent and/or radiation therapy can be varieddepending on the disease being treated and the known effects of thechemotherapeutic agent and/or radiation therapy on that disease. Also,in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents (i.e., antineoplastic agent or radiation) on thepatient, and in view of the observed responses of the disease to theadministered therapeutic agents.

[0088] Also, in general, the HSP90 inhibitor and the chemotherapeuticagent do not have to be administered in the same pharmaceuticalcomposition, and may, because of different physical and chemicalcharacteristics, have to be administered by different routes. Forexample, the HSP90 inhibitor may be administered orally to generate andmaintain good blood levels thereof, while the chemotherapeutic agent maybe administered intravenously. The determination of the mode ofadministration and the advisability of administration, where possible,in the same pharmaceutical composition, is well within the knowledge ofthe skilled clinician. The initial administration can be made accordingto established protocols known in the art, and then, based upon theobserved effects, the dosage, modes of administration and times ofadministration can be modified by the skilled clinician.

[0089] The particular choice of HSP90 inhibitor, and chemotherapeuticagent and/or radiation will depend upon the diagnosis of the attendingphysicians and their judgment of the condition of the patient and theappropriate treatment protocol.

[0090] The HSP90 inhibitor, and chemotherapeutic agent and/or radiationmay be administered concurrently e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,depending upon the nature of the proliferative disease, the condition ofthe patient, and the actual choice of chemotherapeutic agent and/orradiation to be administered in conjunction (i.e., within a singletreatment protocol) with the HSP90 inhibitor.

[0091] If the HSP90 inhibitor, and the chemotherapeutic agent and/orradiation are not administered simultaneously or essentiallysimultaneously, then the initial order of administration of the HSP90inhibitor, and the chemotherapeutic agent and/or radiation, may not beimportant. Thus, the HSP90 inhibitor may be administered first followedby the administration of the chemotherapeutic agent and/or radiation; orthe chemotherapeutic agent and/or radiation may be administered firstfollowed by the administration of the HSP90 inhibitor. This alternateadministration may be repeated during a single treatment protocol. Thedetermination of the order of administration, and the number ofrepetitions of administration of each therapeutic agent during atreatment protocol, is well within the knowledge of the skilledphysician after evaluation of the disease being treated and thecondition of the patient. For example, the chemotherapeutic agent and/orradiation may be administered first, especially if it is a cytotoxicagent, and then the treatment continued with the administration of theHSP90 inhibitor followed, where determined advantageous, by theadministration of the chemotherapeutic agent and/or radiation, and so onuntil the treatment protocol is complete.

[0092] Thus, in accordance with experience and knowledge, the practicingphysician can modify each protocol for the administration of a component(therapeutic agent-i.e., HSP90 inhibitor, chemotherapeutic agent orradiation) of the treatment according to the individual patient's needs,as the treatment proceeds.

[0093] The attending clinician, in judging whether treatment iseffective at the dosage administered, will consider the generalwell-being of the patient as well as more definite signs such as reliefof disease-related symptoms, inhibition of tumor growth, actualshrinkage of the tumor, or inhibition of metastasis. Size of the tumorcan be measured by standard methods such as radiological studies, e.g.,CAT or MRI scan, and successive measurements can be used to judgewhether or not growth of the tumor has been retarded or even reversed.Relief of disease-related symptoms such as pain, and improvement inoverall condition can also be used to help judge effectiveness oftreatment.

[0094] The following examples are not limiting and are merelyrepresentative of various aspects and features of the present invention.All references referred to above and below are incorporated herein byreference.

EXAMPLES Example 1 Effect of Ansamycins on Cells with a Functional RbProtein and Cells Lacking a Functional Rb Protein

[0095] Cell Culture:

[0096] The human breast cancer cell lines MB-MDA 468, MCF7 and BT-549and the colon carcinoma cell line, Colo 205, were obtained from ATCC.Breast cell lines were maintained in DME-F12 media and Colo 205 cells inRPMI; both media were supplemented with 5% fetal calf serum (BRL), 2 mMglutamine and 50 u/ml each of penicillin and streptomycin. All cellswere incubated at 37° C. in 5% CO₂.

[0097] Cells were treated for 24 hours with 250 ng/ml herbimycin A(Gibco) dissolved in DMSO or 435 nM of radicicol (Sigma).

[0098] After treatment the nuclei can Do stained wish ethidium bromideand analyzed by flow cytometry.

[0099] Flow Cytometry:

[0100] Nuclei were isolated for flow cytometry assays stained withethidium bromide and analyzed using a Becton Dickinsonfluorescence-activated cell sorter. Statistical data was obtained usingMulticycle program software.

[0101] Results:

[0102] As shown in FIG. 1, in 12 tumor cell lines examined, ansamycintreatment caused growth arrest in G₁. In tumor cell lines with mutatedRb HA treatment failed to induce a G₁ block but instead resulted to anaccumulation of cells with 4n DNA content (FIG. 1B).

Example 2 Analysis of Cell Arrest in Rb-Negative and Rb-Positive CellsTreated With HSP90 Inhibitors

[0103] Mitotic Index:

[0104] For mitotic indices, cells were typsinized, washed with PBS andfixed with 3% paraformaidehyde in PBS for 20 min. Cells were thenstained with 3 μg/ml bisbenzimide (Hoechst 33258; Sigma) for 15 min andexamined under fluorescence microscopy. Mitosis was scored by thepresence of condensed chromosomes.

[0105] Immunofluorescent Analysis:

[0106] For immunofluorescent analysis, harvested cells were washed withPBS, fixed with methanol for 20 min at −20° C., washed again and blockedfor 30 min with 2% BSA in PBS. Cells were then stained withanti-α-tubulin (Sigma) and anti-centromere protein antibodies(ACA/CREST) (gift of Dr. J. D. Rattner) in 2% BSA PBS for 1 hour.Following 3 washes with 0.5% BSA in PBS, cells were incubated withanti-human FITC conjugated, antimouse rhodamine conjugated antibodies(Molecular Probes) and 2 μg/ml bisbenzimide in 2% BSA in PBS for 45 min.Cells were then washed 4× with 0.5% BSA in PBS, resuspended in PBS andimages captured by confocal microscopy or with a CCD camera. Images werethen processed using Slidebook 3.0 and Adobe Photoshop program software.

[0107] For synchrony experiments, cells were treated with 1 μg/mlaphidicolin (Sigma) for 18 hours, washed and replated in mediacontaining DMSO or HA.

[0108] Results:

[0109] As shown in FIG. 3, α-tubulin staining demonstrates that arrestedcells contained bipolar spindles, indicating that ansamycins do notinterfere with spindle formation. Additionally, in most cells,chromosomes localized both to the poles and within the spindle (FIG.3A): ACA staining revealed paired centromeres on chromosomes at thepoles (FIG. 3B). In 77 chromosomes localized to the poles, 87%, scoredas double dots for ACA staining, indicating that accumulation ofchromosomes at the poles is not the result of premature or incompletesegregation but rather, failure of paired chromatids to assemble to thespindle equator. These data show that HA-treated cells are arrested inpromethaphase and that, in Rb-negative cells, HA induces mitotic arrestby interfering with chromosomal alignment.

Example 3 Measurement of Mitotic Cycline

[0110] Immunoblot Analysis:

[0111] Levels of mitotic cyclin expression and associated kinaseactivities in herbimycin arrested MB-MD468 cells were assessed usingimmunoblot analysis and in vitro kinase assays as described below. Cellscultured with herbimycin were enriched for mitotically arrested cells byusing only the loosely adherent population in which the mitotic indexwas greater than 90%.

[0112] Immunoblot analysis of lysates from cells treated with DMSO,nocodazole or herbimycin were analyzed by Western blot analysis usinganti-cyclin A or anti-B1 antibodies. Treated cells were harvested,washed with PBS and lysed in NP40 lysis buffer (50 mM Tris pH 7.4, 1%NP40,150 mM NaCl, 40 mM NaF. 1 mM Na₃VO₄, 1 mMphenylmethylsulfonlylfluoride, and 10 μg/ml. each of leupeptin,aprotinin and soybean trypsin inhibitor) for 30 min on ice. Lysates werecentrifuged at 15,000×g for 10 min and protein concentration determinedby bicinchoninic acid protein assay (Pierce). Equal amounts of totalprotein were resolved by SDS-PAGE and transferred onto Immobilon PVDFmembranes (Millipore) by electroblotting. Blots were blocked overnightin 5% nonfat milk in TBS-T (0.1% Tween-20 TBS, 10 mM Tris pH 7.4, 150 nMNaCl) at 4° C. and subsequently probed with either anti-cyclin A orcyclin B1 antibodies (Santa Cruz Biotechnology). Following incubationwith HRP-conjugated secondary antibodies, proteins were detected bychemiluminescence (Amersham).

[0113] Immunoprecipitation and in Vitro Kinase Assays:

[0114] For immunoprecipitation, 100 μg of total protein was incubatedwith anticyclin A or anti-cyclin B1 (Santa Cruz) antibodies for 2 hoursat 4° C. and then for 1 hour following the addition of proteinA-Sephaliose. The immune complexes were washed 4× with lysis buffer andboiled in SDS-PAGE sample buffer for 5 min. Following SDS-PAGE, proteinswere transferred onto Immobilon and analyzed by western blotting.

[0115] For in vitro kinase reactions, immune complexes were washed 4×with lysis buffer, 2× with kinase buffer (20 mM Tris pH 7.4, 7.5 mMMgCl₂, 1 mM DTT) and incubated in 40 μl of kinase buffer containing 2 μghistone H1, 10 μCi [γ-³²P] ATP and 300 μM ATP for 10 min at 37° C. Thereaction was stopped by the addition of SDS-PAGE sample buffer andboiled for 5 min. Proteins were resolved on SDS-PAGE, transferred ontoImmobilon and exposed to autoradiography film or phosphoimager screen.Kinase activity was quantitated by FUJIX phosphoimager and MacBASprogram software.

[0116] Results:

[0117] Cyclin B1-associated kinase activity was elevated 5-fold inHA-treated cells when compared to control and was comparable to thatseen in nocodazole-arrested cells (FIG. 4B). Cyclin B1 proteinexpression was also increased in HA-treated cells (FIG. 4B). Incontrast, cyclin A expression and its associated kinase activity wereslightly lower in both HA and nocodazole-aresseted cells compared tothat in control cells (FIG. 4A).

Example 4 Effect of Herbimycin in Cells Expressing Human PapilommaVirus-16 E6 and E7

[0118] Primary human foreskin keratinocytes transfected with HPV-1 6 E6and E7 were provided by Drs. H. Stöppler and R. Schlegel (GeorgetownUniv.) and grown as previously described. Primary human foreskinkeratinocytes or HPV 16 E6/E7 transfected human foreskin keratinoyteswere treated with HA or DMSO for 24 hours and ethidium bromidestained-nuclei analyzed by flow cytometry as described above.

[0119] Results:

[0120] HA caused the majority of primary HFK cells (FIG. 6A) toaccumulate in G₁, in contrast E6/E7 transfectants arrested with 4n DNAcontent (FIG. 6B).

Example 5 Transfection of Rb Gene into Rb-Negative Cells

[0121] Rb Transfection:

[0122] To further confirm that the gene product of the Rb gene permitsprogression through mitosis in the presence of an HSP90 inhibitor,MB-MDA 468 cells were transfected with the plasmid pUHD10-3HGRcontaining full-length 4.7 kb human Rb cDNA. Rb transfectants were grownin DME-F12 media supplemented with 5% fetal calf serum (BRL), 2 mMglutamine and 50 μg/ml each of penicillin and streptomycin and 100 μg/mlhygromycin B (Boehringer Mannheim). To confirm the presence of the Rbgene product Western blot analysis was carried out using anti-Rbantibodies (Pharmingen) as described herein. Vector control, MB-MDA 468,and Rb transfected MB-MDA 468 cells were arrested with aphidicolin asdescribed above. After release from aphidicolin arrest, transfected andnon-transfected MM-MDA 468 cells were cultured in the presence of anansamycin inhibitor as described in Example 1. Cell progression wasmonitored by flow cytometric analysis of ethidium bromide-stained nucleias described above.

[0123] Results:

[0124] When treated with HA, control transfectants (MB-MDA 468 cells)accumulated with 4n DNA content. In contrast, in the Rb transfectants(468-7; 468-19) HA caused an increase in G₁ and had no effect on G₂/M(data not shown). When released from aphidicolin block into HA, Rbtransfectants cycled through mitosis and entered G₁ (FIGS. 5C-5D). Incontrast, when treated with HA after aphidicolin block, MB-MDA 468 cellsfailed to reach G₁ and arrested in mitosis by 12 hours (FIG. 5B).

[0125] All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually. None of thereferences are admitted to be prior art.

[0126] One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

[0127] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention. Thus, such additional embodiments are within the scope of thepresent invention and the following claims.

[0128] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of” and “consisting of” may be replaced witheither of the other two tern's. The terms and expressions which havebeen employed are used as terms of description and not of limitation,and there is no intention that in the use of such terms and expressionsof excluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments, optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by thedescription and the appended claims.

[0129] In addition, where features or aspects of the invention aredescribed in terms of Markush groups or other grouping of alternatives,those skilled in the art will recognize that the invention is alsothereby described in terms of any individual member or subgroup ofmembers of the Markush group or other group.

[0130] Other embodiments are within the following claims.

1. A method for destroying cells that are deficient in retinoblastomagene product, comprising administering to said cells a compound capableof inhibiting HSP90 function.
 2. The method of claim 1, wherein saidcompound is an ansamycin.
 3. The method of claim 2, wherein saidansamycin is selected from the group consisting of geldanamycin, 17-AAG,or herbimycin A.
 4. The method of claim 2, wherein said ansamycin is17-AAG.
 5. The method of claim 1, wherein said compound is radicicol. 6.The method of claim 1, wherein said compound is a synthetic compoundthat binds into the ATP-binding site of a HSP90.
 7. A method of treatingdisorders associated with cells that are deficient in retinoblastomagene product, comprising administering a therapeutically effectiveamount of a compound capable of inhibiting HSP90.
 8. The method of claim7, wherein said disorder is small cell lung cancer.
 9. The method ofclaim 7, wherein said disorder is associated with a viral infection. 10.The method of claim 9, wherein said viral infection is caused by a Humanpapilloma virus.
 11. The method of claim 9, wherein said disorder iscervical cancer.
 12. The method of claim 7, wherein said compound isgiven in combination with another therapy for treating the disorder. 13.The method of claim 7, wherein said compound is an ansamycin.
 14. Themethod claim 13, wherein said ansamycin is selected from the groupconsisting of geldanamycin, 17-AAG, or herbimycin A.
 15. The method ofclaim 13, wherein said ansamycin is 17-AAG
 16. The method of claim 7,wherein said compound is radicicol.
 17. The method of claim 7, whereinsaid compound is a synthetic compound which binds in the ATP-bindingsite of a HSP90.
 18. The methods of claims 1 or 7, wherein said cellsare RB negative.